臺灣博碩士論文加值系統

本研究以SACCHACHITIN（靈芝多醣幾丁質）進行研究，探討其對酪胺酸酶活性的影響。由於之前已證實此材料能有效抑制蛋白質水解酵素（如MMP-1，MMP-8，MMP-9）以及脂肪酶（Lipase），所以本實驗將進一步探討其抑制酪胺酸酶活性之成效。本研究首先探討對抑制蕈酪胺酸酶 （Mushroom tyrosinase）活性的影響性，結果顯示：2.5 mg/ml的抗壞血酸顯示出非常顯著的抑制作用，在不同來源的幾丁聚醣中，只有SACCHACHITIN呈現劑量依存反應（dose-dependent reaction）其IC50約為17.2 mg/ml。接著在細胞模式下，SACCHACHITIN對B16細胞型態和增生沒有不良影響，不具有細胞毒性。再進行B16細胞黑色素含量的分析，加入誘導劑α-MSH濃度為25 nM所增加的百分比率最高，可以提高3.37倍的黑色素含量。不論在無或有誘導劑之下， SACCHACHITIN均可以減少B16細胞產生黑色素，和降低B16細胞之酪胺酸酶活性，呈現劑量依存反應（dose-dependent reaction）。西方點墨法再次確認SACCHACHITIN確實能夠抑制B16細胞中酪胺酸酶蛋白質的表現，尤其是當SACCHACHITIN濃度為2 mg/ml時，效果更加顯著，同時且隨著SACCHACHITIN的微粒化，有增進抑制B16細胞中酪胺酸酶蛋白質表現的結果。

Abstract
SACCHACHITIN was prepared from the waste residue of the fruiting body of Ganoderma taugae, was used in our previous study to inhibit matrix metalloproteinases and lipase. In the present study, SACCHACHITIN was estimated for its effects on the tyrosinase activity and expression. In the mushroom tyrosinase inhibition assay, the results indicated that 2.5 mg/ml of ascorbic acid had strong inhibition of mushroom tyrosinase. In different source of chitin, only SACCHACHITIN showed a potent tyrosinase activity inhibition, IC50 value was 17.2 mg/ml. In the cell culture assay, SACCHACHITIN showed neither morphological alteration nor disorder in proliferation. These evidences elucidated that SACCHACHITIN was not cytotoxic and safe toward the growth of B16 mouse melanoma cells. Use 25nM -MSH induce melanogenesis in B-16 cells could elevate 3.37 times of melanin content. The results of inhibitory effect of SACCHACHITIN with no inducer and with inducer -MSH on melanin biosynthesis and tyrosinase activity were observed in B-16 mouse melanoma cells all in a dose-dependent response reaction.
Western blot was employed to ensure SACCHACHITIN inhibited B16 cells tyrosinase protein expression, and the results indicated that 2.0 mg/ml SACCHACHITIN had obviously inhibitary effect. In the same experiment, SACCHACHITIN in small particle ( average size: 10 μm ) seemed in clined to enhance the inhibition of tyrosinase protein expression.

致謝
中文摘要 1
英文摘要 2
緒 論
壹. 靈芝子實體細胞壁組成SACCHACHITIN（靈芝多醣幾丁質） 3
一、 SACCHACHITIN的來源 3
二、 SACCHACHITIN 的安全性 3
三、 SACCHACHITIN 促進傷口癒合 4
四、 SACCHACHITIN 的抗菌活性 5
五、 SACCHACHITIN 對酵素的抑制作用 6
貳. 酪胺酸酶是調控黑色素形成的關鍵酵素 6
參. 黑色素的形成（Melanogenesis）與調節 8
肆. 皮膚的顏色 10
伍. 人體皮膚的構造與生理功能 12
陸. 常見美白產品的作用機轉 13
柒. 實驗假說與目的 15
實驗流程圖 16
材料與方法 17
一、 儀器設備 17
二、 試藥與溶劑 18
三、 SACCHACHITIN 之萃取與微粒化 20
（1） SACCHACHITIN 之萃取流程 20
（2） SACCHACHITIN 之微粒化 20
四、 蕈酪胺酸酶活性抑制分析（Mushroom tyrosinase inhibition assay） 21
五、 細胞增生與存活分析（Determination of Cell Proliferation and Viability） 21
六、 B16細胞之黑色素含量分析（Melanin content assay of B16 cells） 22
七、 B16細胞沈澱物的分析（B16 Cell-Pellet Assay） 22
八、 B16細胞之酪胺酸酶活性分析（Tyrosinase Activity assay of B16 cells） 22
九、 西方墨點法分析B16老鼠黑色素瘤細胞中酪胺酸酶蛋白質表現 23
結果
1. 蕈酪胺酸酶活性抑制分析（Mushroom Tyrosinase Inhibition Assay）
1-1. 萃取物抑制蕈酪胺酸酶活性分析 26
1-2. SACCHACHITIN 抑制蕈酪胺酸酶活性分析 26
2. 測定萃取物對B16細胞增生分析（Determination of B16 Cells Proliferation and
Viability）
2-1. 抗壞血酸對於B16細胞增生與存活分析 27
2-2. SACCHACHITIN對於B16細胞增生與存活分析 27
2-3. α-MSH對於B16細胞增生與存活分析 28


3. 萃取物對於B16細胞之黑色素含量分析（Melanin content assay of B16 cells）
3-1. α-MSH對於B16細胞之黑色素含量分析 29
3-2-1. 抗壞血酸對於B16細胞之黑色素含量分析 30
3-2-2. 加入誘導劑後（α-MSH）抗壞血酸對於B16細胞之黑色素含量分析 31
3-3-1. SACCHACHITIN對於B16細胞之黑色素含量分析 31
3-3-2. 不同顆粒大小的SACCHACHITIN對於B16細胞之黑色素含量分析 32
3-3-3. 不同顆粒大小SACCHACHITIN在加入黑色素誘導劑後對B16細胞之
黑色素含量分析 33
4. 萃取物對於B16細胞之酪胺酸酶活性影響（Tyrosinase Activity assay of B16 cells）
4-1-1. 抗壞血酸對於B16細胞之酪胺酸酶活性影響 34
4-1-2. 加入誘導劑後（α-MSH）抗壞血酸對於B16細胞之酪胺酸酶活性影響 34
4-2-1. 不同濃度SACCHACHITIN對B16細胞之酪胺酸酶活性分析 35
4-2-2. 加入誘導劑後（α-MSH）不同濃度SACCHACHITIN對於B16細胞之
酪胺酸酶活性影響 35
4-2-3. 不同顆粒大小的SACCHACHITIN對B16細胞之酪胺酸酶活性影響 36
5. 西方墨點法（Western Blots）分析B16老鼠黑色素瘤細胞中酪胺酸酶蛋白質表現
5-1. α-MSH對於B16 細胞之酪胺酸酶蛋白質表現分析 37
5-2. SACCHACHITIN對於B16細胞之酪胺酸酶蛋白質表現分析 37
5-3. 不同顆粒大小SACCHACHITIN對B16細胞之酪胺酸酶蛋白質表現分析
38

6. 不同顆粒大小的SACCHACHITIN對B16細胞型態之影響 38

討 論 39
結論與展望 44
圖 表 46
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